Non-vesicular cationic lipid formulations

ABSTRACT

The present invention relates to a non-vesicular preparation comprising at least one cationic amphiphile in an aqueous environment, its production and use and a cationic liposome suspension obtainable thereof with increase drug trap ratio and its areas of application such as pharmacology and medicine, particularly its use as carrier system for active substances.

The present invention relates to a non-vesicular preparation comprisingat least one cationic amphiphile in an aqueous environment, itsproduction and use and a cationic liposome suspension obtainable thereofwith increased drug trap ratio and its areas of application such aspharmacology and medicine, particularly its use as carrier system foractive substances.

Liposomes play a significant role in medical and pharmaceutical sciencesas drug delivery systems. In a typical application, an active compound,if it is lipopohilic, is encapsulated in the bilayer lipid membrane ofthe liposome or, if it is hydrophilic, is inserted into the aqueouscompartment, in order to have it delivered to a target site.

For the preparation of liposomes a variety of well-known methods areavailable (R. R. C. New (ed.) Liposomes, A Practical Approach, OxfordUniversity Press, Oxford 1990). However, liposomes which comprisewater-soluble compounds, and which fulfil the requirements ofhomogeneity, narrow size distribution with small liposome sizes, as wellas high drug to lipid values are still difficult to achieve. High drugto lipid ratios however, are of particular importance for medicalapplications.

With the usual standard methods for liposome formation, theencapsulation efficacy for water-soluble compounds is low. For example,a compound can be loaded on basis of the well known film method: A thinfilm of lipid on the inner wall of a flask is reconstituted with anaqueous solution, which contains the compound to be encapsulated. Thefraction of the compound which is enclosed in the so-formed liposomescorresponds to the fraction of encapsulated with respect to the totalvolume. Common liposome formulations have concentrations in the rangefrom 10-50 mM with liposome diameters in the range from 100 to 300 nm.For such formulations, the ratio of encapsulated to total volume issmall and therefore the encapsulation efficacy is small. Most of thecompound remains in the free aqueous phase and is usually removed bydialysis. This has the further disadvantage that most of the valuablecompound is lost.

The non-encapsulated compound is removed, since it may cause sideeffects if it is not protected in the liposomal carrier. Further, it mayhave pharmacokinetic characteristics which are different to those of theliposomal drug. In case of targeted delivery by the liposomes, thenon-liposomal fraction of the compound is inactive. For this reasons itis important to minimize the non-liposomal fraction of the drug.

A variety of methods has been described to overcome this intrinsicproblem of encapsulation of compounds in the aqueous compartment ofliposomes. One of it is the active loading technique, which isapplicable to compounds where the membrane permeability can bedifferent, for example as a function of the pH value (R. R. C. New (ed.)Liposomes, A Practical Approach, Oxford University Press, Oxford 1990).In that case, by applying a pH gradient from the inner to the other sideof the liposome, the compound can be trapped in the vesicle. However,these approaches are applicable only to a limited number of suitablemolecules and to particular environmental conditions. Therefore, so farnone of them provided a substantial general breakthrough for liposomalformulations of water soluble compounds.

In WO 96/05808 and WO 99/49716 a method for producing concentrated‘vesicular phospholipid gels’ by using high-pressure homogenisation isdisclosed. These semi-solid phospholipid pastes or -gels with high lipidcontent consist predominantly of vesicular structures (WO 96/05808, WO99/49716 and Brandl 2001 (M. Brandl (2001) Liposomes as drug carriers: atechnological approach, Biotechnology annual review Volume 759-85). WO96/05808 discloses liposome preparations from unilamellar vesicles ofsmall and medium size (100-300 nm), with high/drug ratios of at least20% w/w. However, several disadvantages are linked to that approach: Thepreparation is highly viscous, and re-dispersion is done best underrigorous mechanical stress, such as an oscillating bath mill which is adisadvantage for delicate materials. As well WO 99/49716 refers toliposome gels, with at least 20% of an active compound, wherein thecompound is added to the liposome gel and, by heating or mechanicalstress, the compound is equally distributed inside and outside thevesicles. However, due to the high viscosity of these liposome gels, anddue to the size of the vesicles, sterile filtration, which is animportant step during the formation of pharmaceutical preparations, isnot possible.

Recently it was reported, that cationic liposomes have high affinity toangiogenic blood vessels around a solid tumor (Schmitt-Sody M. et al.(2003) Clin Cancer Res 9, 2335-41), which makes them useful for specifictargeting of a drug to the tumor site (vascular targeting). However, ashas been discussed above, many drugs of interest can partition into theaqueous phase. For liposomal formulations of such compounds a certainfraction is present in the free aqueous phase and thus is inactive withrespect to the targeting capacity of cationic liposomes.

In general, for compounds which have a certain solubility in water orwhich have a high permeability across the membrane, the loading of theliposome with the drug is a problem which has not been sufficientlysolved so far. In all presently available approaches a significantfraction of the compound is not encapsulated. It is not active in thesense of specific targeting of the carrier. It may be removed bydialysis or equivalent techniques, but a significant amount will belost. Another difficulty is, that the encapsulated state is usually anon-equilibrium state, since in the thermodynamic equilibrium thecompound is uniformly distributed. Therefore, depending on the membranepermeability of the compound, during the time between dialysis andapplication further material may be released from the liposome into theaqueous phase.

The problem underlying the present invention was to provide an improveddrug delivery and/or release system with a high drug to lipid ratio,target specificity and sufficient stability for pharmaceuticalapplication.

Thus, the solution to the above problem is achieved according to theinvention by providing the embodiments characterized in the claims.

The invention relates to a non-vesicular preparation comprising at leastone cationic amphiphile in the range of about 10 mM to about 600 mM,preferably of about 25 mM to about 500 mM, more preferably of about 100mM to about 400 mM, and most preferably of about 200 mM to about 300 mM,optionally a further amphiphile in the range of about up to 60 mol %with respect to the total amphiphile concentration and optionally astabilizing agent in the range of about 10 mM to about 600 mM,preferably of about 100 mM to about 500 mM and more preferably of about200 mM to about 400 mM.

Unexpectedly, it was found, that a clear transparent phase which isvirtually free of light scattering particles and which is not adispersion of liposomes or any other particulate dispersion can beobtained if cationic amphiphiles, preferably lipids are mixed in anaqueous phase. This new phase can be obtained with a wide range ofamphiphile concentrations, from about <20 mM up to about >600 mM. Itappears that there is no lower concentration limit, and the highconcentration limit is close to the state of swollen lipid bilayers withno excess of water.

The inventive preparation can be described by being a transparent,isotropic, substantially homogeneous phase which differs in variousfundamental aspects from classical liposome suspensions (FIG. 2). As adirectly visible attribute, liposome suspensions appear white opalescentdue to light scattering from liposome particles. The inventivepreparation, to the contrary, is clear and transparent, i.e., virtuallyno light scattering particles are present. Trials of quasi elastic lightscattering measurements (Zetasizer 3000, Malvern, Herrenberg, Germany)indicate that the scattering intensity is reduced by at least a factorof 300 with respect to liposome suspensions with a mean size of about180 nm. Under usual conditions, liposome suspensions of 1 mMconcentrations give a count rate of about 60 kCps. For the inventivepreparation of DOTAP at 270 mM a count rate of about 40 kCps ismeasured. Virtually no size distribution can be determined and virtuallyno indication for particles >10 nm is found (Malvern Contin analysis).

The particle number can also be deduced from turbidity measurements,which can be performed by UV-vis spectroscopy. In FIG. 5 the UV spectraof a 30 mM DOTAP liposome suspension and of a 270 mM non-vesicularpreparation of DOTAP are shown. As can be seen, the absorption (andtherefore the scattering) is much higher for the liposome suspension asfor the non-vesicular preparation, even though the latter has aconcentration which is about one order of magnitude higher. Comparisonof absorption at a selected wavelength (400 nm) indicates, that themolar scattering of the non vesicular preparation is less than 2% ofthat of the liposome suspension.

As a further characteristic, the inventive preparation shows lowmacroscopic viscosity up to rather high lipid concentrations (>200 mM),i.e., visual inspection suggests a liquid like state, similar to that ofthe aqueous phase since it can be easily extruded through membranes of200 nm pore size (the pore size which is usually used for sterilefiltration). This makes the preparation potentially applicable as aready to use pharmaceutical composition also for applications in whichsterile filtration is demanded, especially if an active compound ispresent. Compared with this, viscosity of liposome suspensions above acertain concentration (>50 mM) are often too high for extrusion andsterile filtration and thus not suitable for pharmaceutical use.

The inventive preparation is remarkably different from formerlydescribed so-called vesicular liposome gels (WO 96/05808 and WO99/49716) since gels are solid-like or semi-solid colloidal structures.The named liposome gels are composed of individual lipid vesicles athigh packing density. In order to allow a component to migrate into alipid vesicle, mechanical agitation or elevated temperature is necessary(WO 99/49716). The inventive preparation however, can be described as ahomogeneous phase wherein no encapsulated or free aqueous phase can bedistinguished. All components in the aqueous phase are free to moveacross the whole volume. If a further component is added, it candistribute across the whole phase and a uniform mixture can be achieved.

The inventive preparation can be transformed into a liposome suspensionby dilution with water or an aqueous solution. Since the inventivepreparation can be also produced at low concentrations, this result wasunexpected. In fact, by the ‘single phase method’ the inventivepreparation can be obtained already at concentrations <25 mM and bysubsequent further solvent evaporation it can be concentrated up to morethan 600 mM without affecting its physical state. (i.e., it continues tobe a clear, transparent phase). It was therefore rather expected thatthe inventive preparation can be diluted without affecting its molecularstate of aggregation. Instead, by the dilution, the molecularorganization changes and liposomes are formed.

So-formed liposomes are preferably in the small to medium size range(30-300 nm) with a narrow size distribution (PI values from sizemeasurements by quasi-elastic light scattering >0.5), which makes themapplicable for pharmaceutical application. Further, entrapment of awater-soluble active compound in the aqueous compartment of theliposomes (formed by dilution of the preparation as disclosed) is afunction of the encapsulated/total volume at the time of liposomeformation. If liposome formation occurs at a concentration which ishigher than that of the final liposome concentration (which is usuallyin the range from 10 to 25 mM), e.g. at a concentration of about 100 mM,the resulting trap rate of the obtainable liposomes is higher as can beachieved if the liposomes are formed directly at a low concentration(for example by reconstitution of a lipid film with an aqueous phasewhich contains the component, see FIG. 3).

Summarizing, the inventive preparation has the following advantages: Itis

-   -   suitable for direct pharmaceutical use    -   suitable for loading an active compound    -   suitable for the preparation of liposomes with a high trap rate        and a narrow size distribution.

The present invention might be characterized more specifically by itsmethod of production. Lipid dispersions in water may exist in a largenumber of different phase and aggregation states, which may bethermodynamically stable or metastable (D. F. Evans, H. Wennerström: TheColloidal Domain: Where Physics Chemistry, Biology and Technology Meet,VHC publishers, Weinheim, 1994). Therefore, by selecting a differentmode of preparation a different type of molecular organization in theresulting phase can be obtained. If that phase state is not thethermodynamically most favourable one, nevertheless it can be stable forlong time periods, particularly long enough to provide sufficient shelflife for production and storage before an application. On the otherhand, a metastable phase may be transformed into a more stable one byapplying a suitable stress to the system.

As an example, a procedure will be given to obtain the inventivepreparation at a molecular composition, for which by using anotherprocedure, classical liposomes are obtained: a 25 mM dispersion of DOTAPin water can be produced as a classical liposome dispersion, for exampleif it is produced by the well-known film method or by ethanol injection.If the dispersion is produced by the subsequently described ‘singlephase evaporation technique’ however, with the identical molecularcomposition, the inventive preparation is obtained. Thethermodynamically less favourable state is hindered from transforminginto the more favourable one by the high energy barrier of such atransition. In order to form or break a liposome, which is the morefavourable thermodynamic state, the lipid bilayer must be disrupted,which requires a significant amount of energy.

In general, the inventive preparation can be obtained by several ways,e.g. by mixing water and an organic solvent, in which the amphiphilesare solubilized. By removing the organic solvent, the inventivepreparation is formed. Any other technique however, well known in theart which permits to obtain a particle free dispersion of lipid in waterby chemical, physical or mechanical means is thereby suitable to producethe inventive preparation. On the other hand all procedures in which therupture of bilayers and subsequent re-fusion to closed vesicle isinvolved, i.e., the procedures which are usually applied for liposomeproduction, like the well know film method or ethanol injection, areless favourable, since these can lead to the formation of vesicles canwhich remain, stable or metastable, in the preparation. Thereforereconstitution of a lipid film to multilamellar vesicles, such asdescribed in WO 96/05808, should be avoided to obtain the inventivepreparation.

The inventive preparation comprises cationic amphiphiles, which areselected from lipids, lysolipids or pegylated lipids having a positivenet charge. The lipid may comprise several, e.g. two hydrocarbon chains,which are not necessarily identical, which are branched or unbranched,saturated or unsaturated with a mean chain length from C12 to C24.Preferred are cationic lipids with at least one tertiary amino orquaternary ammonium group.

Useful lipids for the present invention include:

-   DDAB, dimethyldioctadecyl ammonium bromide;    N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium methylsulfate    (DOTAP); 1,2-diacyloxy-3-trimethylammonium propanes, (including but    not limited to: dioleoyl, dimyristoyl, dilauroyl, dipalmitoyl and    distearoyl; also two different acyl chain can be linked to the    glycerol backbone); N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine    (DODAP); 1,2-diacyloxy-3-dimethylammonium propanes, (including but    not limited to: dioleoyl, dimyristoyl, dilauroyl, dipaimitoyl and    distearoyl; also two different acyl chain can be linked to the    glycerol backbone);    N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride    (DOTMA); 1,2-dialkyloxy-3-dimethylammonium propanes, (including but    not limited to: dioleyl, dimyristyl, dilauryl, dipalmityl and    distearyl; also two different alkyl chain can be linked to the    glycerol backbone); dioctadecylamidoglycylspermine (DOGS);    3-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol);    2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminium    trifluoro-acetate (DOSPA); -alanyl cholesterol; cetyl trimethyl    ammonium bromide (CTAB); diC14-amidine;    N-tert-butyl-N′-tetradecyl-3-tetradecylaminopropionamidine; 14Dea2;    N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride    (TMAG);    O,O′-ditetradecanoyl-N-(trimethylammonioacetyl)diethanolamine    chloride; 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide    (DOSPER);    N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium    iodide;    1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium    chloride derivatives as described by Solodin et al. (1995) Biochem.    43:13537-13544, such as    1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazolinium    chloride (DOTIM),    1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium    chloride (DPTIM), 2,3-dialkyloxypropyl quaternary ammonium compound    derivatives, containing a hydroxyalkyl moiety on the quaternary    amine, as described e.g. by Felgner et al. [Felgner et al. J. Biol.    Chem. 1994, 269, 2550-2561] such as:    1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI),    1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide    (DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium    bromide (DORIE-HP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxybutyl    ammonium bromide (DORIE-HB),    1,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide    (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl    ammonium bromide (DMRIE),    1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide    (DPRIE), 1,2-disteryloxypropyl-3-dimethyl-hydroxyethyl ammonium    bromide (DSRIE); cationic esters of acyl carnitines as reported by    Santaniello et al. [U.S. Pat. No. 5,498,633]; cationic triesters of    phosphatidylcholine, i.e.,    1,2-diacyl-sn-glycerol-3-ethylphosphocholines, where the hydrocarbon    chains can be saturated or unsaturated and branched or non-branched    with a chain length from C₁₂ to C₂₄, the two acyl chains being not    necessarily identical.

In a preferred embodiment the cationic amphiphile is selected from aquaternary ammonium salt such asN-[1-(2,3-diacyloxy)propyl]-N,N,N-trimethyl ammonium, wherein apharmaceutically acceptable counter anion of the quaternary aminocompound is selected from the group consisting of chloride, bromide,fluoride, iodide, nitrate, sulfate, methyl sulfate, phosphate, acetate,benzoate, citrate, glutamate or lactate. Preferably, the cationic lipidsare in the liquid crystalline state at room temperature. Examples arelipids where the hydrocarbon chains contain one or more double bonds,where the hydrocarbon chains are branched, or where any other packingmismatch is given, for example due to different chains. Further, in manycases lipids with chains shorter than C14 fulfil the requirement.

The inventive preparation may comprise at least one further amphiphilein an amount of about 0 to about 60 mol %, preferably of about 20 mol %to about 50 mol % and most preferably of about 30 mol % to about 40 mol% based on the total amphiphile concentration.

The further amphiphiles may have a negative and/or neutral net charge(anionic and/or neutral amphiphile). These can be selected from sterolsor lipids such as cholesterol, phospholipids, lysolipids,lysophospholipids, sphingolipids or pegylated lipids with a negative orneutral net change. Useful anionic and neutral lipids thereby include:Phosphatidic acid, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol (not limited to a specific sugar), fatty acids,sterols containing a carboxylic acid group, cholesterol,1,2-diacyl-sn-glycero-3-phosphoethanolamine, including but not limitedto dioleoyl (DOPE), 1,2-diacyl-glycero-3-phosphocholines, sphingomyelin.The fatty acids linked to the glycerol backbone are not limited to aspecific length or number of double bonds. Phospholipids may also havetwo different fatty acids. Preferably the further lipids are in theliquid crystalline state at room temperature and they are miscible (i.e.a uniform phase can be formed and no phase separation or domainformation occurs) with the used cationic amphiphile, in the ratio asthey are applied.

In a preferred embodiment the neutral amphiphile is phosphatidylcholine.

The preparation may further comprise a stabilizing agent, which ispreferably selected from a sugar or a polyvalent alcohol or acombination thereof such as trehalose, maltose, sucrose, glucose,lactose, dextran, mannitol or sorbitol. In a preferred embodiment thestabilizing agent is trehalose or glucose.

The preparation may further comprise an organic solvent, particularly awater-soluble organic solvent, e.g. ethanol in an amount up to about 5%(v/v). Instead of ethanol other alcohols or organic solvents can be usedas well. For producing a pharmaceutical composition, organic solventswhich are not ethanol may need to be removed. Suitable organic solventsare alcohols, e.g. methanol, ethanol, propanol, isopropanol, or ethyleneglycol, ethers, e.g. tetrahydrofuran or diethylether, or halogenatedhydrocarbons, e.g. chloroform, or mixtures of these solvents.

Unless defined otherwise, all technical and scientific terms used inthis specification shall have the same meaning as commonly understood bypersons of ordinary skill in the art to which the present inventionpertains. “About” in the context of amount values refers to an averagedeviation of maximum +/−20%, preferably +/−10% based on the indicatedvalue. For example, an amount of about 30 mol % cationic lipid refers to30 mol %+/−6 mol % and preferably 30 mol %+/−3 mol % cationic lipid withrespect to the total lipid/amphiphile molarity.

“Amphiphile” refers to a molecule, which consists of a water-soluble(hydrophilic) and an oil-soluble (lipophilic) part. Lipids andphospholipids are the most common representatives of amphiphiles. In thetext, lipid and amphiphile are used synonymously.

“Angiogenesis associated condition” e.g. refers to different types ofcancer, chronic inflammatory diseases, rheumatoid arthritis, dermatitis,psoriasis, wound healing and others.

“Camptothecin” refers to20(S)-Camptothecine(1H-Pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione, 4-ethyl-4-hydroxy-, (S)—), CAS 7689-03-4. ‘Camptothecin’ or‘camptothecin drug’ in the present context includes as well thecarboxylate form of the drug.

“Camptothecin drug” refers to camptothecin itself or a derivativethereof. A camptothecin derivative is obtained by any chemicalderivatization of camptothecin (see structure). A non-limiting list ofpossible camptothecin drugs is given under: http://dtp.nci.nih.gov asfrom Aug. 19, 2002. In the sketch of the molecule, the most frequentderivatization sites are outlined as R₁-R₅.

Structure of camptothecin drugs:

In the following table, typical examples for derivatization at differentsites are listed. Camptothecin may be present as a hydrochloride. Thelactone ring (E-ring) may be seven-membered instead of six-membered(homocamptothecins). Name R1 R2 R3 R4 R5 Camptothecin H H H H H 9-Nitro-H H NO₂ H H camptothecin 9-Amino- H H NH₂ H H camptothecin 10-Hydroxy- HOH H H H camptothecin Topotecan H OH —CH₂—N—(CH₃)₂ H H SN38 H OH HCH₂—CH₃ H Camptosar ®(Irinotecan) H

H CH₂—CH₃ H Lurtotecan ® R1 and R2 is: H H H O—CH2—CH2—O DX-8951f F CH₃R₃ and R₄ is: H —CH2—

Derivatization can influence the properties of CPT to make the moleculemore hydrophilic or more lipophilic, or that the lactone-carboxylateequilibrium is affected. In the context of the application of CPT as ananti-cancer drug, derivatization is intended to maintain or to increaseactivity.

“Cancer” refers to the more common forms of cancers such as bladdercancer, breast cancer, colorectal cancer, endometrial cancer, head andneck cancer, leukaemia, lung cancer, lymphoma, melanoma, non-small-celllung cancer, ovarian cancer, prostate cancer and to childhood cancerssuch as brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma,ependymoma, Ewing's sarcoma/family of tumors, germ cell tumor,extracranial, hodgkin's disease, leukemia, acute lymphoblastic,leukemia, acute myeloid, liver cancer, medulloblastoma, neuroblastoma,non-hodgkin's lymphoma, osteosarcoma/malignant fibrous histiocytoma ofbone, retinoblastoma, rhabdomyosarcoma, soft tissue sarcoma,supratentorial primitive neuroectodermal and pineal tumors, unusualchildhood cancers, visual pathway and hypothalamic glioma, Wilms' Tumorand other childhood kidney tumors and to less common cancers includingacute lymphocytic leukaemia, adult acute myeloid leukaemia, adultnon-hodgkin's lymphoma, brain tumor, cervical cancer, childhood cancers,childhood sarcoma, chronic lymphocytic leukaemia, chronic myeloidleukaemia, esophageal cancer, hairy cell leukaemia, kidney cancer, livercancer, multiple myeloma, neuroblastoma, oral cancer, pancreatic cancer,primary central nervous system lymphoma, skin cancer, small-cell lungcancer.

“Carrier” refers to a diluent, adjuvant, excipient, or vehicle which issuitable for administering a diagnostic or therapeutic agent. The termalso refers to a pharmaceutically acceptable component(s) that contains,complexes or is otherwise associated with an agent to facilitate thetransport of such an agent to its intended target site. Carriers includethose known in the art, such as liposomes, polymers, lipid complexes,serum albumin, antibodies, cyclodextrins and dextrans, chelates, orother supramolecular assemblies.

“Cationic” refers to an agent that has a net positive charge or positivezeta potential under the respective environmental conditions. In thepresent invention, it is referred to environments where the pH ist inthe range between 3 and 9, preferably between 5 and 8.

“Cationic amphiphiles” as used herein refer to cationic lipids asdefined.

“Cationic liposome” refers to a liposome which has a positive netcharge. In the present invention, it is referred to environments wherethe pH is in the range between 3 and 9, preferably between 5 and 8. Thecationic liposomes are prepared from the cationic lipids or amphiphilesthemselves or in admixture with other amphiphiles, particularly neutralor anionic lipids.

“Derivative” refers to a compound derived from some other compound whilemaintaining its general structural features. Derivatives may be obtainedfor example by chemical functionalization or derivatization.

“Drug” as used herein refers to a pharmaceutically acceptablepharmacologically active substance, physiologically active substancesand/or substances for diagnosis use.

“Encapsulation efficiency” refers to the fraction of a compound which isencapsulated into the liposomes of a liposome suspension by a givenmethod.

“Homogenization” refers to a physical process that achieves a uniformdistribution between several components. One example is high-pressurehomogenisation.

“Lipid” in its conventional sense refers to a generic term encompassingfats, lipids, alcohol-ether-soluble constituents of protoplasm, whichare insoluble in water. Lipids are amphiphilic molecules such as fattyacids, steroids, sterols, phospholipids, glycolipids, sulpholipids,aminolipids, or chromolipids. The term encompasses both naturallyoccurring and synthetic lipids. In a more general sense, lipids arecharacterized as amphiphiles, i.e., they are molecules which consist oflipophilic as well as hydrophilic moieties. Preferred lipids inconnection with the present invention comprise at least two alkyl chainswith at least 12 carbon chains and are: steroids and sterol,particularly cholesterol, phospholipids, including phosphatidyl andphosphatidylcholines and phosphatidylethanolamines, and sphingomyelins.Fatty acids could be about 12-24 carbon chains in length, containing upto 6 double bonds, and linked to the backbone. The hydrocarbon chainscan be different (asymmetric), or there may be only 1 fatty acid chainpresent, e.g., lysolecithins. Mixed formulations are also possible,particularly if non-cationic lipids are derived from natural sources,such as lecithins (phosphatidylcholines) purified from egg yolk, bovineheart, brain, or liver, or soybean.

“Uposome” refers to a microscopic spherical membrane-enclosed vesicle(about 50-2000 nm diameter) made artificially in the laboratory. Theterm “liposome” encompasses any compartment enclosed by a lipid bilayer.Liposomes are also referred to as lipid vesicles.

“Lysolipid” refers to a lipid where one fatty acid ester has beencleaved resulting in a glycerol backbone bearing one free hydroxylgroup.

“Lysophospholipid” refers to a phospholipid where one fatty acid esterhas been cleaved resulting in a glycerol backbone bearing one freehydroxyl group.

“Negatively charged lipids” refer to lipids that have a negative netcharge. In the present invention, it is referred to environments wherethe pH is in the range between 3 and 9, preferably between 5 and 8.Examples are phosphatidic acid, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol (not limited to a specificsugar), fatty acids, sterols.

“Neutral lipids” refer to lipids that have a neutral net charge such ascholesterol, 1,2-diacyl-sn-glycero-3-phosphoethanolamine, including butnot limited to dioleoyl (DOPE), 1,2-diacyl-glycero-3-phosphocholines,Sphingomyelin. In the present invention, it is referred to environmentswhere the pH is in the range between 3 and 9, preferably between 5 and8.

“Non-vesicular cationic preparation” as used herein refers to acomposition comprising at least one cationic amphiphile in an aqueousenvironment. The overall net charge of the amphiphiles is positive, alsoif further anionic or neutral amphiphiles are present.

“Particle diameter” refers to the size of a particle. To experimentallydetermine particle diameters, dynamic light scattering (DLS)measurements, using Malvern Zetasizer 1000 or 3000 (Malvern, Herrenberg,Germany) were performed. For quantitative data analysis (determinationof Z(average) and PI were determined, or, additionally, data analysiswith the ‘Contin’ formalism was performed.

“Pegylated lipid” refers to a lipid bearing one ore more polyethyleneglycol residues.

“Pharmaceutical composition” refers to a combination of two or moredifferent materials with superior pharmaceutical properties than arepossessed by either component.

“Phospholipid” refers to a lipid consisting of a glycerol backbone, aphosphate group and one or more fatty acids wich are bound to theglycerol backbone by ester bonds.

“Positively charged Lipids” refer to a synonym for cationic lipids (fordefinition see definition of “cationic lipids”). In the presentinvention, it is referred to environments where the pH is in the rangebetween 3 and 9, preferably between 5 and 8.

“Stabilizing agent” as used herein refers to a compound which is watersoluble and favourable for the stability of the inventive preparation.

“Sterol” refers to a steroid alcohol. Steroids are derived from thecompound called cyclopentanoperhydrophenanthrene. Well-known examples ofsterols include cholesterol, lanosterol, and phytosterol.

“Therapeutic agent” refers to a species that reduces the extent of thepathology of a disease such as cancer. Such a compound may, for example,reduce primary tumor growth and, preferably, the metastatic potential ofa cancer. Alternatively, such a compound may reduce tumor vascularity,for example either by decreasing microvessel size or number or bydecreasing the blood vessel density ratio.

“Virtually free” of a species refers to as not detectable by HPTLG.“Virtually free of liposomes” refers to a state, where the signal from agiven method such as light scattering, which is proportional to theliposome concentration, is less than 5% of the value as it is obtainedin a system which has the same molecular composition but consisting ofliposomes.

The inventive preparation is a substantially homogeneous phasecomprising at least one cationic amphiphile, optionally at least onefurther amphiphile, optionally a stabilizing agent and optionally anactive compound. The active compound can thereby be hydrophilic,lipophilic or amphipathic compound or a mixture of compound and ispreferably selected from a therapeutic or a diagnostic agent.

Preferably, a therapeutic agent is present in the range of about 0.1 mol% to about 20 mol % with respect to the total amphiphile concentrationpreferably in the range of about 1 mol % to about 15 mol % and morepreferably in the range of about 3 mol % to about 10 mol %.

The therapeutically active agent may be selected from ananti-inflammatory drug, an anti-cancer drug, an enzymatic drug, anantibiotic substance, an antioxidant, a hormone drug, an angiogenesisinhibiting agent, a smooth muscle cell-proliferation/migrationinhibitor, a platelet aggregation inhibitor, a release inhibitor for achemical mediator, and a proliferation/migration inhibitor for vascularendothelium. Specific examples are selected from taxanes, from otheragents interacting with microtubuli such as epothilones, discodermolide,laulimalide, isolaulimalide, eleutherobin, colchicines and derivativesthereof, vinca alkaloids such as vinorelbine, from platinum complexessuch as oxaliplatin, from camptothecins, from anthracyclines such asdoxorubicin or from statins (e.g., lovastatin).

More preferably the inventive preparation comprises camptothecin, acamptothecin drug or a derivative thereof in the range of about 0.1 mol% to about 20 mol %, preferably in the range of about 1 mol % to about15 mol % and more preferably in the range of about 3 mol % to about 10mol % with respect to total amphiphile concentration.

In a further embodiment, the active compound is selected from diagnosticagents such as imaging agents, e.g. magnetic resonance imaging agents(gadolinium complexes such as Magnevist, Omniscan and others), X-ray andcomputed tomography contrast agents (compounds with heavy elements witha large number of electrons such as iodine, barium, dysprosium andothers; examples include ionic and non-ionic derivatives of iodinatedbenzoic acid derivatives such as iopamidol and iodixanol, barium sulfateand others), and other agents employed in other imaging modalities(ultrasound, fluorescence, near infrared and others).

Preferably a diagnostic agent such as an imaging agent is present in therange of about 0.1 mol % to about 50 mol %, preferably in the range ofabout 10 mol % to about 50 mol % and more preferably in the range ofabout 30 mol % to about 50 mol % with respect to total amphiphileconcentration.

As has been disclosed above, unexpectedly, after dilution with anaqueous solution, a suspension of liposomes may be obtained from theinventive preparation. Thus, in a further aspect the present inventionrelates to a cationic liposome suspension obtainable from thenon-vesicular preparation as disclosed.

Unexpectedly, so-formed liposomes are characterized by a well-definedsize distribution. For example, after dilution of a preparationcomprising about 280 mM DOTAP and 2.5 mM CPT to a final DOTAPconcentration of 25 mM, size measurement by quasi-elastic lightscattering indicate a Z_(average) of 70 nm and a PI value of 0.4. InFIG. 4, results from analytical ultracentrifugation measurements aregiven. A very narrow size distribution was obtained.

In a preferred embodiment the inventive cationic liposome suspensioncomprises liposomes of a defined size distribution in the range betweenabout 50 nm to about 1000 nm and in a more preferred embodimentliposomes with a size distribution of about 50 nm to about 500 nm,preferably of about 50 nm to about 300 nm. The small liposome size withwell defined size distribution makes the suspension particularlysuitable for direct pharmaceutical administration.

Further, the liposome suspension may comprise the liposomally loadedcompound in a higher amount as can be obtained with methods state of theart, i.e., the liposomes are ‘overloaded’ with the compound. Theliposomes are produced from the inventive preparation by dilution. Themaximum gain which is theoretically obtainable can be estimated on basisof a simple calculation: If the preparation is formed at 100 mM totalamphiphile concentration, and the final liposome concentration is 10 mM,the fraction of free active compound is reduced by a factor of about tencompared with liposome formulations produced by standard techniques suchas lipid film or ethanol injection method.

The present invention is suitable for pharmaceutical application.Accordingly, the present invention provides a pharmaceutical compositioncomprising the inventive preparation or the cationic liposome suspensionas disclosed, optionally together with a pharmaceutically acceptablecarrier, diluent and/or adjuvant.

If a purely water-soluble active agent is present at the time ofdilution, it is enclosed into the aqueous compartment of the liposome toa higher fraction as if liposomes are formed by classical techniques. Ifa water-soluble compound can partition into the membrane bilayer, itstrap rate in the membrane after dilution will be higher than itsequilibrium state at the same concentration. Unexpectedly the release ofsuch a compound from the membrane into the free aqueous phase can occurslowly enough to enable pharmacological administration and thus, theabove described vascular targeting effect can be achieved with higherefficiency as with liposomal formulations disclosed in the prior art.

For many liposomal formulations the hydrophilic compound is releasedfrom the liposome with a certain time constant. This is particularly thecase, if membrane permeability of the compound is high. In many casesthe release is too fast to enable production and storage with sufficientshelf life before administration. It is an advantage of the presentinvention, that the liposome suspension or a pharmaceutical compositionobtainable thereof can be provided directly before use. If the inventivepreparation, already comprising an active compound, is stored in theconcentrated state only a very low fraction is released into the freeaqueous phase, since the relative volume of the aqueous phase is small.If it may not have sufficient shelf life, the inventive preparation andactive compound can be stored separately, and mixed and diluted directlybefore use. In this way, formulations with a lifetime, which is usuallyvery low can be provided for a pharmaceutical application. For example,if an encapsulated water-soluble compound is released from the liposomewithin a time scale of days or even several hours, it cannot be storedbefore application. Even if such a formulation is prepared directlybefore use by a classical method, the non-encapsulated fraction must beremoved in a time consuming procedure. With the inventive approach, thecompound and the concentrated non-vesicular preparation can be mixed,optionally sterile filtrated, and reconstituted to a liposome suspensionwith a high encapsulation ratio directly before pharmaceuticalapplication, and the liposome suspension can be used directly afterdilution. Therefore, even loaded liposome suspensions with a short halflife of hours can be provided for application on a regular basis.

Thus, another aspect of the present invention relates to a kitcomprising the inventive preparation and an aqueous solution of anactive compound as disclosed.

Camptothecin carboxylate is a compound, which is water soluble, but itpartitions in cationic lipid membranes due to favourable interactionswith cationic lipids. In order to maximize the liposomal fraction, it isdesirable to maximize the lipid concentration. However, for practicalapplications, too high liposome concentrations are disadvantageous, forexample due to the high viscosity.

By using a concentrated non-vesicular preparation comprising cationicamphiphiles, preferably lipids and camptothecin, liposomes can beformed, wherein the liposomal fraction corresponds to the concentratedstate directly after dilution, i.e., it is temporarily higher than theequilibrium state after dilution. The equilibrium is reached only afterfew hours, and therefore, if liposomes are prepared from the inventivenon-vesicular preparation and applied directly after dilution, they willhave a higher fraction of liposomal camptothecin and thus a higherefficacy than liposomes in the equilibrium state.

For illustration, in FIG. 4 a liposome suspensions as obtained from theinventive preparation, and a classical liposome suspension, as producedby ethanol injection and extrusion are compared. Both liposomesuspensions comprise 22.5 mM DOTAP and 2.5 mM camptothecin. Using theinventive preparation, a non-vesicular phase comprising 450 mM DOTAP and50 mM camptothecin was diluted to a concentration of 22.5 mM DOTAP. Then10 ml of both suspensions were diluted 1:10, and from the resulting 100ml the free camptothecin was removed by cross-flow filtration. In thecourse of filtration, the aqueous phase comprising all molecularlydissolved compounds can pass across the membrane. The filtrate wasaliquoted in volumina of 5 ml and the amount of free CPT was determinedby UV-vis spectroscopy. In FIG. 4, the absorption in the filtrate isshown for the liposomes as obtained from the inventive preparationdirectly after dilution, the same after two days and, for comparison,the results of a normal liposome suspension. As can be seen, directlyafter dilution the fraction of free CPT is by about a factor of twolower than after two days. The values for the free camptothecin aftertwo days are still slightly lower that with the classically producedliposomes which might indicate that the equilibrium was still notreached. Generally, with the DOTAP/camptothecin system, the equilibriumis reached after several hours.

The inventive non-vesicular preparation can be produced by a variety ofmethods, such as outlined in the experimental descriptions.

In a further embodiment, the present invention relates to a method ofproducing the non-vesicular preparation comprising cationic amphiphilesas disclosed. As has been outlined, the mode of preparation isfundamental to achieve the inventive preparation. For one and the samemolecular composition, several metastable phase and aggregation statescan occur. Even though these states are thermodynamically metastable,they may be stable in a certain time scale and thus stable enough forproduction and storage with sufficient shelf life for pharmaceuticalapplications. By application of external stress, which can be byaddition of a component, change of pH, mechanical stress, heating or anyother environmental condition, one phase may be transformed intoanother, thermodynamically more favourable one. On the other hand, inorder to keep the system in a certain metatstable phase, it is preferredto avoid such stress.

For production of the inventive preparation preferably but notexclusively at low lipid concentrations (<100 mM) it is favourable torun through a state of a homogeneous lipid solution, for example as amixture of ethanol and water. Such a preparation can be obtained e.g. bysimple mixing an ethanolic lipid solution (about 1 mM to less than about100 mM) with water or an aqueous solution, optionally comprising furthercomponents. Ethanol and optionally partly water is subsequently removedby evaporation and a clear dispersion of lipid in the aqueous phase isobtained (“single phase evaporation method”). The evaporation can occurup to any value with respect to the initial volume, provided there isexcess water left in the preparation.

More specifically, the cationic lipid concentration, preferably DOTAP inenthanol ca be from about 0.5 mM to about 50 mM, more preferably fromabout 1 mM to about 25 mM. The ethanol to water ratio can be in therange from about 1:20 up to about 20:1, preferably from about 1:10 up toabout 10:1 and more preferably from about 1:5 up to about 5:1. The finalconcentration can be any concentration below swollen lipid bilayers withno excess of water, more preferably from about 100 mM to about 600 mM,more preferably from about 200 to about 400 mM.

Instead of lipids amphiphiles as defined may be used and instead ofethanol any suitable organic solvent which is miscible in water such asmethanol, ethanol, propanol, isopropanol, ethylene glycol,tetrahydrofuran, chloroform or diethylether or a mixture of these.

With this procedure, no liposomes are formed. Even though a liposomesuspension may be thermodynamically more favourable, the eventualformation of liposomes is avoided since the energy barrier of formationof closed bilayer vesicle is too high if no sufficient mechanical,thermal or other stress is applied.

To the contrary, in standard liposome preparation procedures a step inwhich mechanical, chemical or other stress is applied to the system inorder to provide sufficient energy to rupture the bilayer membrane toform the closed vesicles. For example, in the film method this is doneby shaking the thin film of swollen lipid bilayers with water, and inethanol injection by the fast dilution of the highly concentratedethanol solution in water.

Another possibility for the formation of the inventive preparation,particularly at high concentration (>100 mM) is high pressurehomogenisation. Dry amphiphiles, preferably lipids and the aqueous phaseare added to the homogenizer without further treatment. Particularly, itis not necessary and not desirable to run through a step of amultilamellar liposome suspension, such as in WO99/49716 and WO96/05808disclosed. Thus, it is necessary to initially avoid any kind of stressin order to avoid the formation of liposomes.

It is a further object of the present invention referring to a method ofproducing the non-vesicular preparation comprising at least one cationicamphiphile, comprising the steps of

-   -   (a) providing said cationic amphiphile, optionally a further        amphiphile, optionally a stabilizing agent, optionally an active        compound and an aqueous phase and    -   (b) subjecting the components of step a) to conditions so that        an isotropic, transparent and substantially homogeneous        preparation is formed.

Step b) therein may comprise the ‘single phase evaporation’ or highpressure homogenisation method.

Preferably, the non-vesicular preparation is prepared by mixing asolution of amphiphiles in an organic solvent with an aqueous phase andsubsequently removing the organic solvent and optionally water to thedesired final concentration (FIG. 1). In this way, the inventivepreparation can be obtained at concentrations of up to the limit ofswollen lipid bilayers, i.e., when no additional water except of thatbinding to the lipid headgroups is present.

However, any other technique suitable for the formation of a uniformparticle free state can be used for producing the inventive preparation,for example such as given in (D. F. Evans, H. Wennerström: The ColloidalDomain: Where Physics Chemistry, Biology and Technology Meet, VHCpublishers, Weinheim, 1994)

As has been disclosed above, the inventive preparation may furthercomprise an active compound. Advantageously, the active compound can besimply either mixed with the amphiphiles for producing the presentpreparation if it is lipophilic, or it can be in the aqueous phase, ifit is water soluble. Alternatively, the active compound can be added toan already formed preparation. If an active compound, dissolved inwater, is added to the already formed inventive preparation, it mayfreely distribute across the whole phase. A lipophilic compound may beadded in dry form and further high pressure homogenisation cycles areapplied for homogeneous distribution in the lipid phase.

Since the inventive preparation is not organized in defined closedvesicles, the homogeneous distribution of the added compound is greatlyfacilitated. Each added compound can distribute homogeneously in thewhole phase, and after dilution the active compound is finallyencapsulated or inserted into the liposomal membrane. The fraction ofthe active compound, which is loaded into the liposome is thereby higheras if the formulation was prepared directly at low lipid concentrationby a standard liposome forming technique as has been outlined above.Thus, liposomal formulations comprising an active compound can beprepared, wherein the liposomal encapsulated fraction of thewater-soluble active compound is increased with respect to theequilibrium state.

The inventive preparation comprising cationic lipids and an activecompound can be taken without further dilution as a ready to usepharmaceutical preparation. Its low viscosity up to high concentrationenables sterile filtration or extrusion through membranes of definedpore size such as with 100 nm or 200 nm pores, which is a prerequisitefor in vivo applications.

As has been disclosed above, the present invention is suitable for thepreparation of a medicament or a diagnostic formulation. Thus, it is afurther object of the present invention that a preparation, a suspensionor a pharmaceutical composition as disclosed can be used for thepreparation of a medicament or a diagnostic formulation, particularlyfor the preparation of a medicament or a diagnostic formulation usefulfor an angiogenesis associated condition such as an angiogenesisassociated disease.

An angiogenesis associated disease is dependent on blood supply. Thelocal interruption of the vasculature will produce an avalanche of celldeath. The vascular endothelium is in direct contact with the blood. Itis contemplated that a variety of diseases can be prevented and treatedwith the foregoing methods and compositions. In a preferred embodiment,a preparation, a liposome suspension or a pharmaceutical composition asprovided by the present invention may be useful for preventing and/ortreating a disease such as cancer, a variety of inflammatory diseases,diabetic retinopathy, rheumatoid arthritis, inflammation, dermatitispsoriasis, stomach ulcers, macular degeneration, hematogenous and solidtumors. In a further preferred embodiment, preparations and compositionsof the present invention can be applied for producing a medicament forpreventing and/or treating solid tumors and their metastases such asbladder, brain, breast, cervical, colorectal, endometrial, head and neckor kidney cancer, leukemia, liver or lung cancer, lymphoma, melanoma,non-small-cell lung, ovarian, pancreatic or prostate cancer.

The preparation of the present invention may be applied directly orafter dilution by injection (e.g. s.c., i.m., i.p.) or implantation. Itis also possible to place it into body cavities or to apply it topicallyonto mucosa, the cornea, or parts of the skin. Thus preparation thusserves as a carrier of the active compound and is responsible for themodified or controlled release of the active compound. Upon transferinto a freely flowing liposome suspension. This suspension may beapplied directly by injection (e.g. s.c., i.m., i.p.) or implantation.It is also possible to place it into body cavities or to apply ittopically onto mucosa, the cornea, or parts of the skin. The entrappingliposomes lead to a distribution of the active substance carried by theliposomes in the body, which distribution selectively effects a high andlong lasting concentration of the active compound at the target site,such is an activated endothelial cell, and thus to an improvement of theeffect or to an improvement of the ratio of effect and side effect, orof the therapeutic or diagnostic index.

Figure Legends:

FIG. 1 Scheme for producing the inventive preparation by the singlephase solvent evaporation: A diluted solution of (cationic) amphiphiles,preferably lipids and an aqueous solution comprising other components(optionally an active compound) are mixed to form a uniform phase. Theorganic solvent, preferably ethanol and, optionally, part of the waterare evaporated until the desired concentration is reached. Thepreparation remains as a clear transparent non-vesicular phase. Afterdilution of the concentrated preparation, liposomes are formed.

FIG. 2 Concentrated preparation containing DOTAP in water at aconcentration of about 250 mg/g (w/w). The preparation is water-clearand liquid like.

FIG. 3 Measurements of free camptothecin (CPT) in different liposomeformulations. A non-vesicular preparation of 450 mM DOTAP and 50 mM CPTwas diluted to a 23.5 mM DOTAP and 2.5 mM CPT liposome suspension.Directly after liposome formation 10 ml of the suspension were furtherdiluted 1:10 and cross-flow filtration was performed. Aliquots of 5 mlof the filtrate were taken and UV-vis measurements were preformed todetermine free CPT. In the graph the absorption at 369 nm is shown. Formthe same 23.5 mM DOTAP liposome suspension, further 10 ml were dilutedafter two days, when the system was expected to be at equilibrium. Ascan be seen, in that case the release is about twice the value asdirectly after dilution. For comparison, a 23.5 mM DOTAP and 2.5 mM CPTliposome suspension was produced directly by ethanol injection. 10 ml ofthe extruded (200 nm) liposome formulation were diluted 1:10 andinvestigated the same way. As can be seen, the values for the free CPTare in the same range as for the suspension from dilution of thenon-vesicular preparation after two days.

FIG. 4 Analytical ultracentrifugation measurements for determining thesize distribution in liposome formulations. Measurements were performedwith 2.5 mM DOTAP and 0.25 mM CPT each. In the upper graph the resultsfrom the measurement of classical liposome formulation as prepared byethanol injection and extrusion (UF60) to a total concentration of 25 mMare shown. For the measurement the sample was diluted 1:10. The lowergraph gives the results from a measurement with liposomes as obtainedfrom a non-vesicular preparation at a total concentration of 500 mM(UF62) after dilution of 1:200. The size distribution of the sample fromdilution of the non vesicular preparation is rather narrow and evenbetter defined that the one of the extruded liposomes.

FIG, 5 UV-Vis spectroscopy measurements comparing the turbidity ofliposome suspensions and the inventive non-vesicular preparation. 30 mMDOTAP liposomes (extruded at 200 nm) and a non-vesicular preparation of270 mM DOTAP were measured. The absorption from the liposome suspensionis much higher that that of the non-vesicular preparation, event thoughthe latter is almost by a factor of ten more concentrated. Quantitativeanalysis (400 nm) indicates that the molar absorption (due toscattering) of the liposome suspension is more than 50 times higher thanthat of the non-vesicular preparation.

The following examples should be illustrative only but are not meant tobe limiting to the scope of the invention. Other generic and specificconfigurations will be apparent to those skilled in the art.

EXAMPLES Example 1

A: Non-Vesicular Preparation of DOTAP in Water at High Concentration(Single Phase Evaporation)

33 ml of an ethanolic DOTAP solution, c=6 mM and 10 ml of a 0.5% aqueoussolution of trehalose were mixed in a round flask. A clear solution wasobtained. The solvent was evaporated at 40° C. at a pressure of 100 mbaruntil the weight of the solution in the flask was 690 mg. Theconcentrate was a clear homogeneous phase, without indication for thepresence of scattering particles. Density of the preparation was about 1g/ml, the resulting DOTAP concentration was about 290 mM and theresulting trehalose concentration was about 7%.

B: Formation of a Liposome Suspension by Dilution

The concentrated preparation of part A was diluted with about 7 ml of10% aqueous trehalose solution to a final concentration of about 25 mMDOTAP. After dilution the clear phase transformed into an opalescentliposome suspension. The size of the liposomes was measured by quasielastic light scattering measurements (Zetasizer 300, Malvern,Herrenbereg, Germany), Z_(ave) 152 nm.

Example 2

Non-Vesicular Preparation of DOTAP at Various Concentrations in theRange from 25 mM to 400 mM (Single Phase Evaporation)

All preparations were formed using a solution of DOTAP (DOTAP-CI) inethanol, c=25 mM and a solution of 10% trehalose in water. For theproduction of the DOTAP preparations with c=25 mM, 100 mM, 200 mM, 300mM und 400 mM the equivalent volumina which are necessary to obtain thedesired final concentrations and aq. trehalose solutions and water weremixed, such as given in the table.

From the solutions solvent was evaporated until a final volume of about0.5 ml was obtained. All concentrates were present as water-clearphases. c (mM) V_(Dotap.) (ml) V_(Trehalose) (ml) V_(H2O) (μl) 25 0.50.5 22 100 2.0 0.5 88 200 4.0 0.5 176 300 6.0 0.5 264 400 8.0 0.5 352

Example 3

Non-Vesicular Preparation of DOTAP in Water at High Concentration (HighPressure Homogenization)

To 8.13 g of DOTAP methyl sulfate 35 ml of water was added. The mixturewas transferred into the pressure chamber of a high pressurehomogenizer. At 750 bar and room temperature, the suspension washomogenized ten times to result in ˜40 ml of a transparent gel-like 300mM formulation.

Example 4

Non-Vesicular Preparation of DOTAP and a Gd Complex in Water at HighConcentration (High Pressure Homogenization)

The High Pressure Homogenizer (Gaulin Micron LAB 40) holds 40 ml ofsample volume. A sample of 36 ml of 0.5 M Gd complex (Omniscan) and 4.65g of DOTAP methyl sulfate are suspended in the pressure chamber. Thehomogenisation procedure (room temperature, 750 bar) is repeatedten-fold to yield the respective material. The experiment is performedwith two DOTAP concentrations, 150 and 300 mM. DOTAP Appearance of GdVolume [mM] Homogenate Stability [mM] increase 150 homogenous fluid noprecipitation at room 9 3.4 temperature 300 homogenous fluid noprecipitation at room 17 2.3 temperature, viscid after dialysis

After homogenisation a homogenous fluid preparation is obtained andextruded through a polycarbonate membrane with 200 nm pore size. Theobtained preparation is dialyzed four times against 5% glucose to removethe non-entrapped contrast agent Omniscan. During dialysis the volume ofthe solution in the dialysis tube increases between 2.3 and 3.4 fold.This increase is taken into account to establish the labellingefficiency. The 300 mM solution turns into a viscous non-vesicular phaseduring this dialysis. The encapsulation efficiency after dialysis is6.1% for 150 mM DOTAP and 7.8% for 300 mM DOTAP.

Example 5

A: Concentrated Non-Vesicular DOTAP/CPT Preparation: DOTAP 500 mM, CPT50 mM (Single Phase Evaporation)

Ethanolic solution of DOTAP (6 mM) was added to an aqueous solution ofCPT-carboxylate (c=2 mM) in 0.5% trehalose with 1% Tris/HCl-buffer, pH7.5. The solvent was evaporated (30° C. and 25 mbar) to a totalconcentration of 500 mM DOTAP and 50 mM camptothecin.

B: Formation of a DOTAP/CPT Liposome Suspension by Dilution andDetermination of the Overloading Directly After Dilution

The clear concentrated preparation of part A was diluted to a DOTAPconcentration of 1 mM (1:500). After dilution an opalescent liposomessuspension was formed.

The fraction of free, non-liposomal, CPT was determined by ‘cross flowfiltration’ across a membrane of 50 kDa MWCO. Free CPT was determineddirectly after dilution and after two days. After dilution the fractionof free CPT was 10% and two days later it was 20%. It is assumed, thatthe state after two days is the equilibrium state. This indicates, thatthe fraction of free CPT was reduced by a factor of two directly afterdilution.

Example 6

Adding of CPT to a Pre-Formed Non-Vesicular Concentrated DOTAPPreparation

To 5 ml of a 280 mM non-vesicular preparation of DOTAP in water asobtained from high pressure homogenization (see Example 3) 5 ml of asolution of a 14 mM solution of CPT carboxylate in water was added. Aclear, slightly yellowish phase was obtained.

1 ml of the preparation were diluted with a 10 mM Tris/HCl buffer, pH7.5 to a final concentration of 15 mM.

Example 7

Tolerability of Liposomes from a Non Vesicular DOTAP/CP Preparation inMice.

A non-vesicular preparation, DOTAP 450 mM camptothecin 25 mM, wasreconstituted with an aqueous solution of 10% trehalose to a liposomesuspension of about 25 mM (dilution 1:20). Directly after dilution, themice were treated with a singe injection of 5 μmol/g. The injectionswere well tolerated, no adverse effects were observed.

Example 8

Human Therapy Treatment Protocols

This example is concerned with human treatment protocols using thepreparations and suspensions disclosed. Treatment will be of use fordiagnosing and/or treating various human conditions and disordersassociated with enhanced angiogenic activity. It is considered to beparticularly useful in anti-tumor therapy, for example, in treatingpatients with solid tumors and hematological malignancies or in therapyagainst a variety of chronic inflammatory diseases such as psoriasis.

A feature of the invention is that several classes of diseases and/orabnormalities are treated without directly treating the tissue involvedin the abnormality e.g., by inhibiting angiogenesis the blood supply toa tumor is cut off and the tumor is killed without directly treating thetumor cells in any manner.

Methods of treating such patients using lipid:drug complexes havealready been formulated. It is contemplated that such methods may bestraightforwardly adapted for use with the method described herein. Asdiscussed above, other therapeutic agents could be administered eithersimultaneously or at distinct times. One may therefore employ either apre-mixed pharmacological composition or “cocktail” of the therapeuticagents, or alternatively, employ distinct aliquots of the agents fromseparate containers.

The various elements of conducting a clinical trial, including patienttreatment and monitoring, will be known to those of skill in the art inlight of the present disclosure.

For regulatory approval purposes, it is contemplated that patientschosen for a study would have failed to respond to at least one courseof conventional therapy and would have objectively measurable disease asdetermined by physical examination, laboratory techniques, orradiographic procedures. Such patients would also have no history ofcardiac or renal disease and any chemotherapy should be stopped at least2 weeks before entry into the study.

The required application volume is calculated from the patient's bodyweight and the dose schedule. Prior to application, the formulation canbe reconstituted in an aqueous solution. Again, the required applicationvolume is calculated from the patient's body weight and the doseschedule.

The disclosed formulations may be administered over a short infusiontime. The infusion given at any dose level should be dependent upon thetoxicity achieved after each. Hence, if Grade II toxicity was reachedafter any single infusion, or at a particular period of time for asteady rate infusion, further doses should be withheld or the steadyrate infusion stopped unless toxicity improved. Increasing doses shouldbe administered to groups of patients until approximately 60% ofpatients showed unacceptable Grade III or IV toxicity in any category.Doses that are ⅔ of this value would be defined as the safe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals of about 3-4weeks later. Laboratory tests should include complete blood counts,serum creatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein.

Clinical responses may be defined by acceptable measure or changes inlaboratory values e.g. tumormarkers. For example, a complete responsemay be defined by the disappearance of all measurable disease for atleast a month. Whereas a partial response may be defined by a 50% orgreater reduction.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Moreover, for human administration, preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biologics standards.

Administration and Dosing

The present invention includes a method of delivery of apharmaceutically effective amount of the inventive preparation orliposome suspension obtainable thereof comprising an active compound toan angiogenic vascular target site of a subject in need thereof. A“subject in need thereof” thereby refers to a mammal, e.g. a human.

The route of administration comprises peritoneal, parenteral or topicadministration and the formulations are easily administered in a varietyof dosage forms such as implantation depots, injectable solutions andthe like. For use with the present invention the term “pharmacologicallyeffective amount” of a compound administered to a subject in needthereof (which may be any animal with a circulatory system withendothelial cells which undergo angiogenesis) will vary depending on awide range of factors. For example, it would be necessary to providesubstantially larger doses to humans than to smaller animal. The amountof the compound will depend upon the size, age, sex, weight, andcondition of the patient as well as the potency of the substance beingadministered. Having indicated that there is considerable variability interms of dosing, it is believed that those skilled in the art can, usingthe present disclosure, readily determine appropriate dosing by firstadministering extremely small amounts and incrementally increasing thedose until the desired results are obtained. Although the amount of thedose will vary greatly based on factors as described above, in general,the present invention makes it possible to administer substantiallysmaller amounts of any substance as compared with delivery systems whichtarget the surrounding tissue e.g., target the tumor cells themselves.

The pharmaceutically effective amount of a therapeutic agent asdisclosed herein depends on the kind and the type of action of theagent. For the examples mentioned here, it is within the range of about0.1 to about 20 mg/kg in humans.

The pharmaceutically effective amount of a diagnostic agent as disclosedherein depends on the type of diagnostic agent. The exact dose dependson the molecular weight of the compound, and on the type and theintensity of the signal to be detected. For the examples as given here(fluorescein as fluorescence dye, gadolinium complexes as MRI markers),the applied dose may range from about 0.1 to 20 mg/kg. Most frequentdoses are in the order of about 5 mg/kg.

1-22. (canceled)
 23. A non-vesicular preparation comprising at least onecationic amphiphile in a concentration of about 10 mM to about 600 mMwith a mean chain length from C12 to C24, optionally at least onefurther amphiphile of up to about 60 mol % based on the total amphiphileconcentration and optionally at least one stabilizing agent in aconcentration of about 10 mM to about 600 mM in an aqueous phase,wherein said preparation is characterized by being transparent,isotropic and substantially homogeneous.
 24. The preparation of claim23, comprising at least one cationic amphiphile in a concentration ofabout 25 mM to about 500 mM, preferably in a concentration of aboutabout 100 mM to about 400 mM and most preferably in a concentration ofabout 200 mM to about 300 mM.
 25. The preparation of claim 23,comprising a stabilizing agent in a concentration of about 100 mM toabout 500 mM, preferably in a concentration of about about 200 mM toabout 400 mM.
 26. The preparation of claim 23, wherein said cationicamphiphile is selected from lipids, lysolipids, pegylated lipids havinga positive net charge.
 27. The preparation of claim 26, wherein saidcationic amphiphile is selected from cationic lipids with at least onetertiary amino or quaternary ammonium group such asN-[1-(2,3-diacyloxy)propyl]-N,N-dimethylamine orN-[1-(2,3-diacyloxy)propyl]-N,N,N-trimethyl ammonium.
 28. Thepreparation of claim 23, wherein said further amphiphile has a negativeor a neutral net charge.
 29. The preparation of claim 23, wherein saidfurther amphiphile is selected from sterols or lipids such ascholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipidsor pegylated lipids with a negative or neutral net change.
 30. Thepreparation of claim 27, wherein the neutral amphiphile isdiacylphosphatidylcholine.
 31. The preparation of claim 23, wherein saidstabilizing agent is selected from a sugar or an alcohol or acombination thereof such as trehalose, maltose, sucrose, glucose,lactose, dextran, mannitol or sorbitol.
 32. The preparation of claim 31,wherein said stabilizing agent is trehalose or glucose.
 33. Thepreparation of claim 23, further comprising an active compound, whereinsaid active compound may be hydrophilic, hydrophobic or amphipathic. 34.The preparation of claim 33, wherein said compound is a therapeuticagent, preferably camptothecin or a derivative thereof, a taxane or another microtubuli interacting agent such as an epothilone,discodermolide, laulimalide, isolaulimalide, eleutherobin, colchicineand/or a derivative thereof, a vinca alkaloid such as vinorelbine, aplatinum complex such as oxaliplatin, an anthracycline such asdoxorubicin or a statin (e.g., lovastatin) and more preferablycamptothecin or a derivative thereof in its carboxylate form.
 35. Thepreparation of claim 34, wherein said therapeutic agent is in the rangeof about 0.1 mol % to about 20 mol %, preferably in the range of about 1mol % to about 15 mol % and more preferably in the range of about 3 mol% to about 10 mol % based on the total amphiphile concentration.
 36. Thepreparation of claim 33, wherein said compound is a diagnostic agent,preferably an imaging agent.
 37. The preparation of claim 36, whereinsaid diagnostic agent is in the range of about 0.1 mol % to about 50 mol%, preferably in the range of about 10 mol % to about 50 mol % and morepreferably in the range of about 30 mol % to about 50 mol % based on thetotal amphiphile concentration.
 38. A method of producing a liposomesuspension comprising using a preparation of claim 23 to form a liposomesuspension.
 39. A method of producing a liposome suspension from thepreparation of claim 23 by diluting said preparation with an aqueoussolution.
 40. A Pharmaceutical composition comprising the preparation ofclaim 23, optionally together with a pharmaceutically acceptablecarrier, diluent and/or adjuvant
 41. A method of preparing a medicamentor a diagnostic formulation comprising using a preparation of claim 23to produce a medicament or diagnostic formulation.
 42. A method oftreating angiogenesis associated condition such as cancer, chronic oracute inflammatory diseases, rheumatoid arthritis, dermatitis, psoriasisor wound healing comprising administering a pharmaceutical compositionof claim
 40. 43. A method of producing the non-vesicular preparation ofclaim 23, comprising: (a) providing i) said cationic amphiphile,optionally said further amphiphile, optionally said stabilizing agent,optionally said active compound, and ii) an aqueous phase; and (b)dispersing the components of i) in said aqueous phase of ii).
 44. Themethod of claim 43, comprising: (a) providing i) said cationicamphiphile, optionally said further amphiphile, optionally saidstabilizing agent, and ii) an aqueous solution; (b) dispersing thecomponents of i) in said aqueous phase of ii); and (c) adding an activeagent to the dispersion of step (b).
 45. The method of claim 43, whereinstep (b) comprises a single phase evaporation or high pressurehomogenisation method.
 46. A method of producing the non-vesicularpreparation of claim 23, comprising: a) providing said cationicamphiphile, optionally said further amphiphile, optionally saidstabilizing agent, optionally said active compound and an aqueous phase;and b) subjecting the components of step a) to conditions so that anisotropic, transparent and substantially homogenous preparation isformed, wherein step b) comprises a single phase evaporation or highpressure homogenisation method.